Streptomyces are producers of a wide variety of secondary metabolites, including most of the commercial antibiotics. Because of this, considerable effort has been invested in developing gene cloning techniques for Streptomyces. Procedures for the efficient introduction of DNA into Streptomyces by polyethylene glycol (PEG) mediated transformation have been developed. Vectors have been constructed which include phages, high copy number plasmids, low copy number plasmids and E. coli-Streptomyces shuttle vectors. Numerous drug resistance genes have been cloned from Streptomyces species and several of these resistance genes have been incorporated into vectors as selectable markers. A review of current vectors for use in Streptomyces is Hutchinson, Applied Biochemistry and Biotechnology 16 pg 169-190 (1988). In many cases, genes for the production of secondary metabolites and genes encoding for resistance have been found to be clustered. Thus one strategy for cloning genes in a pathway has been to isolate a drug-resistance gene and then test the adjacent DNA for other genes for that particular antibiotic. Examples of biosynthetic genes clustered near a drug resistance gene include actinorhodin (Malpartida and Hopwood, Nature 309 pg 462 (1984)), tetracenomycin C (Motamedi and Hutchinson, Proc. Natl. Acad. Sci. USA 84 pg 4445-4449 (1987)), and bialaphos (Murakami et al, Mol. Gen. Genet. 205 p 42-50 (1986 ), EP 173,327). EP 204,549 exploits the clustering of drug-resistance genes and biosynthetic genes and claims a method for isolating antibiotic genes by using a easily isolated drug-resistance gene. Patent publication wo87/03907 discloses a method for isolating polyketide antibiotics using cloned genes for polyketide synthase. This application also discloses the cloning of genes involved in milbemycin biosynthesis, a compound structurally related to the avermectins. Another strategy for cloning genes for the biosynthesis of commercially important compounds has been complementation of mutants. A library of DNA from a producing organism is introduced into a nonproducing mutant and the transformants are screened for the production of the compound. This approach has also identified gene clusters involved in antibiotic production, in some cases all the genes for the production of several antibiotics have been cloned. In addition to the three examples above, other examples of cloned Streptomyces genes involved in antibiotic biosynthesis include tylosin (Fishman et. al., Proc. Natl. Acad. Sci. USA, 84 pg 8248-8252 (1987), undecylprodigiosin (Feitelson, et al., J. Gen. Micro. 131 pg 2431-2441 (1985), methylenomycin (Chater and Bruton, EMBO J 4 pg 1893-1897 (1985), nosiheptide (JP 8636216) and Cephamycin C (Chen et al., Bio/Technology 6 pg 1222-1224 (1988), JP 8667043). In several cases new analogs of antibiotics have been produced by the introduction of cloned genes into other Streptomyces (Floss, Biotechnology 5 pg 111-115 (1987), Hopwood et al., Nature 314 pg 642-644 (1985)). In other cases the introduction of extra copies of biosynthetic genes into the original producing organism has resulted in increased titer of the antibiotic. EP 238323 discloses the process of introducing a gene for the rate limiting enzyme into the producing organism to increase titer of the antibiotic.
Streptomyces avermitilis produces avermectins, a series of 8 related compounds with potent anthelmintic and insecticidal activity (U.S. Pat. Nos. 4,310,519 and 4,429,042). A semisynthetic derivative of avermectin, ivermectin, is a commercially important anthelmintic. U.S. Pat. No. 4,310,519 describes a mutant of S. avermitilis which lacks the furan ring of the natural avermectins. Schulman et al., J. antibiot. 38 pg 1494-1498 (1985) describes a mutant, Agly-1, which produces avermectin aglycones A1a and A2a. Ruby et al., Proceedings of the 6.sup.th International Symposium on the Biology of Actinomycetes, G. Szabo, S. Biro, M. Goodfellow (eds.), p.279-280 (1985) and Schulman et al., Antimicr. Agents and Chemother. 31 pg 744-747 (1987) describe 2 classes of S. avermitilis mutants, one class is defective in O-methylation at C-5 and the other class is defective in O-methylation at C-3" and C-3'. EP 276103 describes a mutant of S. avermitilis defective in branch chain fatty acid dehydrogenase. EP 276131 describes a S. avermitilis mutant defective in C-5, C-3", and C-3' O-methylation. Ikeda et al., J. Bacteriol. 169 pg 5615-5621 (1987), have described the isolation and genetic analysis of two classes of S. avermitilis mutants. AveA mutants were defective in avermectin aglycone formation and AveB mutants failed to synthesize or attach the oleandrose moiety to avermectin aglycone. They obtained genetic evidence that the two classes of mutations are linked. This application describes the cloning of genes required for the biosynthesis of avermectins. Other microorganisms that produce avermectin-like-compounds are S. hygroscopicus, S. cyanogrieseus and S. thermoarchaenosis. Such microorganisms may be subjected to the same procedures as are described herein for S. avermitilis.